Probiotic Confection and Lipid Compositions

ABSTRACT

The present application relates to probiotic confection-based compositions comprising lactic acid-producing bacteria and oil-based compositions comprising the same.

RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 15/090,086, filed Apr.4, 2016, which is a continuation of U.S. Ser. No. 13/086,675, filed Apr.14, 2011, now U.S. Pat. No. 9,301,982, issued Apr. 5, 2016, claiming thebenefit of priority to U.S. Provisional Patent Application No.61/390,355, filed Oct. 6, 2010 and U.S. Provisional Patent ApplicationNo. 61/323,914, filed on Apr. 14, 2010, the entire contents of each ofwhich are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present application relates to probiotic confection and lipidcompositions comprising lactic acid-producing bacteria.

BACKGROUND OF THE INVENTION

The gastrointestinal microflora plays a number of vital roles inmaintaining gastrointestinal tract function and overall physiologicalhealth. The growth and metabolism of the many individual bacterialspecies inhabiting the gastrointestinal tract depend primarily upon thesubstrates available to them, most of which are derived from the diet.These findings have led to attempts to modify the composition andmetabolic activities of the bacterial community through diet, primarilywith probiotics, which are live microbial food supplements.

Probiotic organisms are non-pathogenic, non-toxigenic, retain viabilityduring storage, and typically survive passage through the stomach andsmall intestine. Since probiotics do not generally permanently colonizethe host, they need to be ingested regularly for health promotingproperties to persist.

SUMMARY OF THE INVENTION

The invention is based on the discovery that lactic acid-producingbacteria, particularly Bacillus species, remain viable and retain theirbeneficial probiotic properties in/on confection-based compositions aswell as lipid or oil-based compositions. Accordingly, the inventiondescribes probiotic confection-based compositions and seafood/fish oilsoft-gels or capsules. Specifically, the invention provides an isolatedBacillus coagulans in such compositions. The compositions are suitablefor human or animal consumption.

The invention provides probiotic confection-based compositionscomprising a confection and an isolated Bacillus coagulans bacterium.The Bacillus coagulans bacterium is coated on the exterior surface ofthe confection. Alternatively, the Bacillus coagulans bacterium isinside the confection itself. For example, the bacterium is incorporatedthroughout the confection. Optionally, the composition further comprisesa granulated or powder sugar coating or dusting on the exterior surfaceof the confection. For example, a sugar-sanded jelly confection ischaracterized by a flexible candy base structure and a sugar sandinglayer or coating that comprises B. coagulans spores or vegetative cellsin an admixture with a granulated or powdered sugar or other sweetener.

Suitable confections include hard sweets, fudge, toffee, liquorice,chocolate, jelly candy, marshmallow, and marzipan. Preferably, the jellycandy is a gelatin-based gummi candy. Exemplary gummi candies includegummi bears, gummi worms, gummi frogs, gummi hamburgers, gummi cherries,gummi soda bottles, gummi sharks, gummi army men, gummi hippopotami,gummi lobsters, gummi watermelons, gummi octopuses, gummi apples, gummipeaches, and gummi oranges. Preferably, the probiotic confection-basedcomposition is a gummi bear with isolated Bacillus coagulans coated onthe external surface. The terms “gummi” and “gummy” are usedinterchangeably herein.

In one aspect, the isolated Bacillus coagulans comprise between about0.01% to about 50% by weight of the confection-based composition.Optionally, the isolated Bacillus coagulans comprise between about 0.01%and about 10% by weight of the confection-based composition. Preferably,the isolated Bacillus coagulans comprise between about 0.01% and about5% by weight of the confection -based composition, e.g., about 1%, about2%, about 3%, about 4%, or about 5% by weight of the confection-basedcomposition.

The invention also provides bacterial species including Bacilluscoagulans, e.g., Bacillus coagulans hammer, preferably Bacilluscoagulans hammer strain Accession No. ATCC 31284, or one or more strainsderived from Bacillus coagulans hammer strain Accession No. ATCC 31284(e.g., ATCC Numbers: GBI-20, ATCC Designation Number PTA-6085; GBI-30 orBC³⁰, ATCC Designation Number PTA-6086; and GBI-40, ATCC DesignationNumber PTA-6087; see U.S. Pat. No. 6,849,256 to Farmer).

Optionally, the isolated Bacillus coagulans is in the form of a spore.Alternatively, the isolated Bacillus coagulans is in the form of avegetative cell. In another aspect, the isolated Bacillus coagulans isin the form of a mixture of vegetative cells and spores. The Bacilluscoagulans is predominantly in spore form, e.g., about 75%, about 80%,about 85%, about 90%, about 95%, about 99%, or about 100% spores.Alternatively, the Bacillus coagulans is predominantly in vegetativeform, e.g., about 75%, about 80%, about 85%, about 90%, about 95%, about99%, or about 100% vegetative cells.

The invention provides compositions comprising a dry mix forconfection-based compositions comprising sugar and an isolated Bacilluscoagulans bacterium. The dry mix is between 1% and 50% Bacilluscoagulans bacterium, e.g., about 5%, about 10%, about 15%, about 20%,about 25%, about 35%, about 45%, or about 50% Bacillus coagulansbacterium. Preferably, the dry mix is about 15% Bacillus coagulansbacterium. For example, about 100 pounds of dry mix contains about 15pounds of Bacillus coagulans bacterium and about 85 pounds of sugar.

The dry mix is between about 1% and about 50% by weight of theconfection-based composition, e.g., about 1% to about 20%, about 5% toabout 15%; about 6%, about 7%, about 8%, about 9%, or about 10% byweight of the confection-based composition. For example, a 3 gramconfection-based composition contains about 7% dry mix by weight of theconfection-based composition. A 3.8 to 4 gram confection-basedcomposition contains about 8-9% dry mix by weight of theconfection-based composition.

The invention also provides methods of making a probioticconfection-based composition. First, a confection (e.g., a gummi bear)is provided and heated to about 100° C. to make the confection “sticky”.Subsequently, isolated Bacillus coagulans bacterium and sugar areapplied to an external surface of the confection, thereby making aprobiotic confection-based composition. Preferably, the confection is agummi bear. The isolated Bacillus coagulans comprise between 1% and 10%by weight of the confection-based composition. In one aspect, theisolated Bacillus coagulans is Bacillus coagulans hammer strainAccession No. ATCC 31284. The isolated Bacillus coagulans is selectedfrom the group consisting of GBI-30 strain (ATCC Designation NumberPTA-6086), GBI-20 strain (ATCC Designation Number PTA-6085), and GBI-40strain (ATCC Designation Number PTA-6087). Optionally, the isolatedBacillus coagulans is in the form of a spore. Alternatively, theisolated Bacillus coagulans is in the form of a vegetative cell. Inanother aspect, the isolated Bacillus coagulans is in the form of amixture of vegetative cells and spores.

Bacillus coagulans bacteria are included in the confection-basedcompositions of this invention. Bacterial species include Bacilluscoagulans, e.g., Bacillus coagulans hammer, preferably Bacilluscoagulans hammer strain Accession No. ATCC 31284, or one or more strainsderived from Bacillus coagulans hammer strain Accession No. ATCC 31284(e.g., ATCC Numbers: GBI-20, ATCC Designation Number PTA-6085; GBI-30 orBC³⁰, ATCC Designation Number PTA-6086; and GBI-40, ATCC DesignationNumber PTA-6087; see U.S. Pat. No. 6,849,256 to Farmer).

The invention also provides a probiotic seafood/fish oil-basedcomposition comprising fish oil and an isolated Bacillus coagulansbacterium. For example, the fish oil, e.g., salmon, cod (e.g., codliver) contains omega-3 fatty acids. Alternatively, the oil is omega-3fatty acid krill oil. The oil comprises eicosapentaenoic acid ordocosahexaenoic acid. The composition is encapsulated in as soft-shelledcapsule or a soft gelatin capsule. Alternatively, the composition is agelatin-based gummi candy. In one aspect, the isolated Bacilluscoagulans comprise between 0.01% and 10% by weight of the composition,e.g., about 1% to about 10%; about 2% to about 9%; or about 5% to about8% by weight of the composition.

In some cases, the isolated Bacillus coagulans is in the form of amixture of vegetative cells and spores. Preferably, the isolatedBacillus coagulans is in the form of a spore. More preferably, thebacterium is present as at least 90% spores, e.g., 95%, 98%, or 99%spores. The isolated Bacillus coagulans is selected from the groupconsisting of GBI-30 strain (ATCC Designation Number PTA-6086), GBI-20strain (ATCC Designation Number PTA-6085), and GBI-40 strain (ATCCDesignation Number PTA-6087).

The Bacillus coagulans Hammer strains of the invention arenon-pathogenic and generally regarded as safe for use in human nutrition(i.e., GRAS classification) by the U.S. Federal Drug Administration(FDA) and the U.S. Department of Agriculture (USDA), and by thoseskilled in the art. Furthermore, the Bacillus coagulans Hammer strainsof the invention germinate at or below human body temperature, renderingthem useful as probiotics. Many Bacillus coagulans strains outside theHammer group have mostly industrial applications, little or nonutritional benefit, and environmental contaminants that have not beenevaluated for safety. Moreover, many other non-Hammer strains ofBacillus coagulans grow optimally at temperatures that exceed human bodytemperature and, thus, do not germinate efficiently in the human body.Such strains are less or not suitable as probiotics for humanconsumption.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims. Unless otherwise defined, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Althoughmethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the present invention,suitable methods and materials are described below. All publications,patent applications, patents, Genbank/NCBI accession numbers, and otherreferences mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION OF THE INVENTION

Probiotic organisms are non-pathogenic, non-toxigenic, retain viabilityduring storage, and survive passage through the stomach and smallintestine. Non-pathogenic lactic acid-producing bacteria (i.e., “lacticacid bacteria”), such as the exemplary Bacillus coagulans, remain viableand retain their beneficial probiotic properties in confection-basedcompositions, such as those prepared in boiling water. Specifically, theprobiotic organisms described herein, e.g., Bacillus coagulans strainGBI-30 or BC³⁰, ATCC Designation Number PTA-6086, survive the harshmanufacturing processes of the confection-based compositions describedbelow. Probiotic lactic acid-producing bacteria

A probiotic lactic acid-producing bacteria suitable for use in themethods and compositions of the invention produces acid and isnon-pathogenic. Purified and/or isolated Bacillus coagulans isparticularly useful as a probiotic in the compositions described herein.By “purified” or “substantially purified” is meant a Bacillus coagulansbacterium that is substantially free of contaminating microorganisms orother macromolecules, e.g., polysaccharides, nucleic acids, or proteins.

The confection-based compositions include a lactic acid-producingbacteria, such as a spore-forming Bacillus species, such as B.coagulans. Preferably, the spore-forming Bacillus species of theinvention is B. coagulans Hammer. There are many suitable bacteriaidentified as described herein, although the invention is not limited tocurrently known bacterial species insofar as the purposes and objectivesof the bacteria is described. The property of acid production isimportant to the effectiveness of the probiotic lactic acid-producingbacteria of this invention.

Exemplary methods and compositions are described herein using Bacilluscoagulans as a probiotic. Purified and/or isolated Bacillus coagulans isparticularly useful as a probiotic in confection-based compositions.Probiotic B. coagulans is non-pathogenic and is generally regarded assafe (i.e., GRAS classification) by the U.S. Federal Drug Administration(FDA) and the U.S. Department of Agriculture (USDA), and by thoseskilled in the art.

Bacillus coagulans is a non-pathogenic gram positive spore-formingbacteria that produces L(+) lactic acid (dextrorotatory) in fermentationconditions. It has been isolated from natural sources, such asheat-treated soil samples inoculated into nutrient medium (Bergey'sManual off Systemic Bacteriology, Vol. 2, Sneath, P.H.A., et al., eds.,Williams & Wilkins, Baltimore, MD, 1986). Purified B. coagulans strainshave served as a source of enzymes including endonucleases (e.g., U.S.Pat. No. 5,200,336); amylase (U.S. Pat. No. 4,980,180); lactase (U.S.Pat. No. 4,323,651); and cyclo-malto-dextrin glucano-transferase (U.S.Pat. No. 5,102,800). B. coagulans has been used to produce lactic acid(U.S. Pat. No. 5,079,164). A strain of B. coagulans (referred to as L.sporogenes; Sakaguti & Nakayama (ATCC 31284)) has been combined withother lactic acid producing bacteria and B. natto to produce a fermentedfood product from steamed soybeans (U.S. Pat. No. 4,110,477).

Bacterial species include Bacillus coagulans, e.g., Bacillus coagulanshammer, preferably Bacillus coagulans hammer strain Accession No. ATCC31284, or one or more strains derived from Bacillus coagulans hammerstrain Accession No. ATCC 31284 (e.g., ATCC Numbers: GBI-20, ATCCDesignation Number PTA-6085; GBI-30 (BC³⁰), ATCC Designation NumberPTA-6086; and GBI-40, ATCC Designation Number PTA-6087; see U.S. Pat.No. 6,849,256 to Farmer).

Bacillus coagulans was previously mis-characterized as a Lactobacillusand labeled as Lactobacillus sporogenes (Nakamura et al. 1988. Int. J.Syst. Bacteriol. 38: 63-73). However, initial classification wasincorrect because Bacillus coagulans produces spores and excretesL(+)-lactic acid through metabolism. Both of these characteristicsprovide key features to the utility of Bacillus coagulans. Thesedevelopmental and metabolic aspects required that the bacterium beclassified as a lactic acid Bacillus. In addition, it is not generallyappreciated that classic Lactobacillus species are unsuitable forcolonization of the gut due to their instability in the harsh (i.e.,acidic) pH environment of the bile, particularly human bile. Bycontrast, Bacillus coagulans is able to survive and colonize thegastrointestinal tract in the bile environment and even grown in thislow pH range.

Probiotic Activity of Bacillus Coagulans

It is well-documented clinically that many species of bacterial, mycoticand yeast pathogens possess the ability to cause a variety ofgastrointestinal disorders including, but not limited to: disruption ofnormal gastrointestinal biochemical function, necrosis ofgastrointestinal tissues, and disruption of the bioabsorption ofnutrients, and like conditions. The probiotic microorganism-containingcompositions described herein inhibit these pathogens. Thus, thecompositions are useful in the prophylactic or therapeutic treatment ofconditions associated with infection by these aforementioned pathogens.The probiotic confection-based compositions of the invention are alsoused in the methods described herein for boosting the immune system.

In one aspect, a Bacillus coagulans strain is included in thecomposition in the form of vegetative cells. In another aspect, theBacillus coagulans strain is included in the composition in the form ofspores. The invention also provides for including the Bacillus coagulansstrain in the composition in the form of a powder, a dried cell mass, astabilized paste, or a stabilized gel.

Because Bacillus spores are heat and pressure-resistant and can bestored as a dry powder, they are particularly useful for formulationinto and manufacture of products such as the various confection-basedcompositions described herein. A Bacillus species is well suited for thepresent invention, particularly species having the ability to formspores which are relatively resistant to heat and other conditions,making them ideal for storage (shelf-life) in product formulations,e.g., confection-based compositions. Due to the shelf-stable propertiesof the Bacillus coagulans strains described herein, e.g., Bacilluscoagulans strain GBI-30 or BC³⁰, ATCC Designation Number PTA-6086, theproduct formulations of the invention are not confined to a refrigeratorand may be stored at room temperature.

The Bacillus coagulans of the invention survives storage (shelf-life)from about 12 days to about 2 years; from about 1 month to about 18months; from about 3 months to about 1 year; or from about 6 months toabout 9 months.

The probiotic organisms described herein, e.g., Bacillus coagulansstrain GBI-30 or BC³⁰, ATCC Designation Number PTA-6086, promotedigestive and oral health and support the immune system. The ability ofBacillus coagulans to inhibit various bacterial pathogens wasquantitatively ascertained by use of an in vitro assay. This assay ispart of a standardized bacterial pathogen screen (developed by the U.S.Food and Drug Administration(FDA)) and is commercially available onsolid support disks (DIFCO® BACTROL® Antibiotic Disks). To perform theassay, potato-dextrose plates (DIFCO®) were initially prepared usingstandard procedures. The plates were then individually inoculated withthe bacteria (approximately 1.5×10⁶ CFU) to be tested so as to form aconfluent bacterial bed.

Inhibition of microorganisms (e.g. gastrointestinal pathogens) byBacillus coagulans was subsequently ascertained by placing approximately1.8×10⁶ CFU of Bacillus coagulans in 10 μl of broth or buffer, directlyin the center of the potato-dextrose plate with one test locus beingapproximately 8 mm in diameter per plate. A minimum of three test lociwere used for each assay. The negative control consisted of a 10 μlvolume of a sterile saline solution, whereas the positive controlconsisted of a 1 μl volume of glutaraldehyde. The plates were thenincubated for approximately about 18 hr at 30° C., at which time thezones of inhibition were measured. As designated herein, “excellentinhibition” means the zone was 10 mm or greater in diameter; and “goodinhibition” means the zone was greater than 2 mm in diameter but lessthan 10 mm in diameter.

As expected, no “inhibition” was seen with the negative, saline control,and excellent “inhibition” (approximately 16.2 mm diameter; average ofthree tests) was seen with the positive, glutaraldehyde control. For theenteric microorganisms tested, the following inhibition by Bacilluscoagulans was found: (i) Clostridium species—excellent inhibition; (ii)Escherichia coli—excellent inhibition; (iii) Clostridiumspecies—excellent inhibition, where the zone of inhibition wasconsistently greater than 15 mm in diameter. Similarly, excellentinhibition was also seen for the opportunistic pathogens Pseudornonasaeruginosa, and Staphylococcus aureus. Pathogenic enteric bacteria whichwere inhibited by Bacillus coagulans activity include, but are notlimited to: Staphylococcus aureus; Staphylococcus epidermidis;Streptococcus pyogenes; Pseudomonas aeruginosa; Escherichia coli(enterohemorragic species); numerous Clostridium species (e.g.,Clostridium perfingens, Clostridium botulinum, Clostridium tributrycum,Clostridium sporogenes, and the like); Gardnereia vaginails;Proponbacterium aenes; Aeromonas hydrophia; Aspergillus species; Proteusspecies; and Klebsiella species.

Micro-Encapsulation

In one aspect, the lactic-acid producing bacteria are incorporated intoa microcapsule coating prior to addition to the confection-basedcomposition, using any micro-encapsulation process well-known in theart. The isolated Bacillus coagulans are packaged, or encapsulated,within another material in order to protect the bacteria from thesurrounding environment. The capsules of the invention range in sizefrom one-thousandth of a millimeter to seven millimeters. The internalingredients of the microcapsule are released from their shells invarious ways, including mechanical rupture of the capsule wall,dissolution of the wall, melting of the wall and diffusion through thewall. Thus, micro-encapsulation provides additional protection to theisolated Bacillus bacterium during heat processing of theconfection-based compositions of the invention. Physical methods ofmicro-encapsulation include pan coating, air-suspension coating,centrifugal extrusion, vibrational nozzle, and spray-drying. Chemicalmethods of micro-encapsulation include interfacial polymerization,in-situ polymerization, and matrix polymerization.

Alternatively, the lactic-acid producing bacteria is added to theconfection-based composition without micro-encapsulation.

Probiotic Confection-Based Compositions

The invention is directed to the surprising discovery that lacticacid-producing bacteria, particularly Bacillus species, remain viableand retain their beneficial probiotic properties in confection-basedcompositions. The confection compositions are suitable for human oranimal consumption. In one aspect, the confection-based compositions areadministered to children under 18 years of age, e.g., under 15 years ofage, under 10 years of age, or under 5 years of age. Alternatively, theconfection-based compositions are administered to children and adults ofall ages.

Confectionery includes food items that are rich in sugar or artificialsweeteners, any one or type of which is called a “confection”. The words“candy” or “sweets” are also used for the term “confectionery”. Candy ismade by dissolving sugar in water or milk to form a syrup, which isboiled until it reaches the desired concentration or starts tocaramelize. The type of candy depends on the ingredients and how longthe mixture is boiled, while the final texture of candy depends on thesugar concentration. As the syrup is heated, it boils, water evaporates,the sugar concentration increases, and the boiling point rises. Thus,boiling temperature corresponds to a particular sugar concentration. Ingeneral, higher temperatures and greater sugar concentrations result inhard, brittle candies, while lower temperatures result in softercandies. Candy names come from the process used to test the syrup beforethermometers became affordable: a small spoonful of syrup was droppedinto cold water, and the characteristics of the resulting lump wereevaluated to determine the concentration of the syrup. Long strings ofhardened sugar indicate “thread” stage, while a smooth lump indicates“ball” stages, with the corresponding hardness described. The “crack”stages are indicated by a ball of candy so brittle that the rapidcooling from the water literally causes it to crack. Candy comes in anendless variety of textures from soft and chewy to hard and brittle.

There are a variety of categories and types of confectionery. Hardsweets are based on sugars cooked to the hard-crack stage, includingsuckers, lollipops, jawbreakers (or gobstoppers), lemon drops,peppermint drops and disks, candy canes, rock candy, etc. Hard sweetsalso include candies often mixed with nuts, such as brittle. Otherscontain flavorings including coffee, such as Kopiko. Fudge is aconfection of milk and sugar boiled to the soft-ball stage. Toffee (orTaffy or Tuffy) is based on sugars cooked to the soft-ball stage andthen pulled to create an elastic texture. Tablet is a crumbly milk-basedsoft and hard candy, based on sugars cooked to the soft-ball stage, andcomes in several forms, such as wafers and heart shapes. Liquorice,which contains extract of the liquorice root, is chewier and moreresilient than gum/gelatin candies, but still designed for swallowing.Other types of confection include chocolates, marshmallow, marzipan, anddivinity. Jelly candies include those based on sugar and starch, pectin,gum, or gelatin, e.g., jelly beans, gumdrops, jujubes, cola bottles, andgummies.

Suitable gummi confections include bears, rings, worms, frogs, snakes,hamburgers, cherries, sharks, penguins, hippos, lobsters, octopuses,apples, peaches, oranges, and spiders. Suitable gummi bear sizes rangefrom the standard candy size (or smaller), to gummi bears that weighseveral kilograms. Gummi confections come in a variety of flavors,including raspberry, orange, strawberry, pineapple, and lemon.

Traditional gummi confection (e.g., gummi bears) is made from sugar,glucose syrup, starch, flavoring, food coloring, citric acid, andgelatin. Suitable gelling agents and hydrocolloids can be selected byone of ordinary skill in the art. Examples include gums, carrageenan,gelatin, pectin, high methoxy pectin, alginates, and agar. One ofordinary skill in the art can select a suitable gelling agent orhydrocolloid depending on the desired final texture of the starch moldedpiece. There are some gummi confections made with pectin or starchinstead of gelatin, making them suitable for vegetarians. An exemplaryorganic gummi confection is made with most all natural ingredients, suchas organic tapioca syrup, organic evaporated cane juice, gelatin,organic grape juice concentrate, citric acid, lactic acid, ascorbicacid, colors added (black, carrot juice concentrate, turmeric, annatto),natural flavors, organic sunflower oil, and carnauba wax.

Large sour gummi bears are larger and flatter than traditional gummibears, have a softer texture, and include fumaric acid or other acidingredients to produce a sour flavor. Sour “gummies” are produced byforming a sweet, flavored, and chewy core and subsequently dusting theexterior with a food acid, such as citric acid. The gelling ingredientin the core of these products is ordinarily gelatin or pectin. Theacidic exterior is applied by use of a wetting agent or food adhesive.Some manufacturers produce sour bears with a different texture, based onstarch instead of gelatin. Typically, starch produces a shorter (cleanerbite, less chewy) texture than gelatin.

Confection-based compositions, such as those described herein, are madefrom a variety of ingredients known to those skilled in the art. Theconfection-based compositions are prepared by combining confectioningredients and a liquid, e.g., water or milk. In one aspect, thecomposition is prepared by combining confection ingredients and aliquid, and heating the resulting combination. Optionally, thecombination is heated (heat-processed) using applied heat, a flame, or amicrowave. The confection-based composition is boiled in hot water,e.g., stovetop boiling, addition of boiling water to a container, ormicrowaving the confection-based composition along with water. In oneaspect, boiling water (about 100° C.). is added to a combination ofconfection ingredients and Bacillus coagulans bacteria.

Mass production of gummi confection (e.g., gummi bears) includes mixingthe gummi confection ingredients and pouring the resulting mixture intomany starched-lined (e.g., corn starch-lined) trays/molds. The cornstarch prevents the gummy bears from sticking to the mold and lets themrelease easily once they are set. First, the desired character molds arecreated and, if necessary, duplicated with a machine. Optionally, starchpowder is applied to the character molds. Gummi confection ingredients,such as sugar, glucose syrup, gelatin, and water are mixed together andheated. In one aspect, the ingredients are mixed with colors and flavorsthat give the bears their signature look and taste. The molten gelatinmixture is poured into the molds and allowed to cool and set prior topackaging or consumption. Preferably, the gummi confection issubsequently heated and placed in a large drum tumbler to apply acomposition of isolated Bacillus coagulans and a sweetener (e.g., asugar).

More specifically, as described in WO/2009/102575, production of gummiconfection includes the following. A colloid batch and a puree batch areformed and combined with corn syrup and sugar to form a base slurry. Thecolloid batch comprises a solution of the gelling agent in water at alevel of from 5 to 15% by weight of the gelling agent, more preferablyfrom 7 to 12% of the gelling agent based on the total weight of thecolloid batch. The colloid batch is held at a temperature of 170 to 190°F. The puree batch preferably comprises water, fruit puree and/or highfructose corn syrup or other sweeteners, thin boiling starch, and sodiumcitrate. It is held at a temperature of from 65 to 75° F. Preferably,the fruit puree has a Brix of from 10 to 45, more preferably from 25 to40. Optionally, the puree batch includes a plurality of fruit purees.The fruit puree comprises a typical fruit puree, a fruit juice, or afruit powder. The puree batch comprises from 30 to 40% by weight water,from 0 to 40% by weight fruit puree, from 0 to 40% by weight highfructose corn syrup, from 25 to 35% by weight thin boiling starch, andfrom 0.0 to 2.0% by weight sodium citrate. In a mixing kettle from 25 to40% by weight of additional corn syrup is combined with from 15 to 40%by weight of fine granulated sugar, from 10 to 15% by weight of thecolloid batch and from 20 to 30% by weight of the puree batch to formthe base slurry. Preferably, the corn syrup is approximately 42 DE cornsyrup, however, as would be understood by one of ordinary skill in theart other DE corn syrups could be used. The base slurry components arecompletely mixed and held at 130 to 150° F. in a holding tank.

The base slurry is then cooked to bring the Brix to from 70 to 85 Brix,more preferably to a Brix of from 75 to 80. In one embodiment the baseslurry is passed through a coil cooker and heated to a temperature offrom 250 to 325° F. to cook it. Other cooking methods could be used aswill be understood by one of ordinary skill in the art. The cooked baseslurry is preferably subjected to vacuum to further increase the Brixinto the desired range. The cooked base slurry is held at approximately200° F. until used. An acidulant solution is preferably added along withcolor and flavor to the cooked base slurry just prior to deposition inthe starch molds. In one aspect, the acidulant solution comprisesascorbic acid present in an amount of from 15 to 20% by weight, citricacid present in an amount of from 10 to 20% by weight, and malic acidpresent in an amount of from 5 to 10% by weight with the remaindercomprising water. As would be understood by one of ordinary skill in theart, other edible acids could be used in place of or in addition tothose listed. In one aspect, 95 to 97% by weight of cooked base slurryis combined with from 2 to 3% by weight of the acidulant solution andthe remainder comprises flavors and colors. Optionally, the acidulantsolution is used to bring the pH of the base slurry to from 2.6 to 3.2.One of ordinary skill in the art would have no difficulty selectingsuitable colors and flavors. The combined mixture is then deposited intostarch molds, e.g., using a Mogul starch molding machine. Such starchmolding machines are well known by those of ordinary skill in the art.In one aspect, from 0.3 to 3 grams of the base slurry is deposited intoeach mold cavity. The starch trays with deposited base slurry aretransferred to a drying room where there are held for 12 to 48 hours.Optionally, the trays are first held at a temperature of from 130 to150° F. for from 10 to 15 hours, and then cooled to 70 to 80° F. andheld at that temperature for from 6 to 12 hours. The gelled starchmolded food pieces are then removed from the trays, the starch isrecycled.

Preferably, the confections of the invention further comprise asweetener (e.g., a granulated or powder sugar) coating on the exteriorsurface of the confection. The sweeteners can comprise one or moremonosaccharides or disaccharides. Examples include sugar, sucrose,invert sugar, dextrose, lactose. honey, malt syrup, malt syrup solids.maltose, fructose. granular fructose, maple syrup, rice syrup, ricesyrup solids, sorghum syrup, refiners syrup, corn syrup, corn syrupsolids, high fructose corn syrup, molasses, or combinations thereof.Sanding sugar comprises cane sugar, beet sugar, date sugar, sucanat,granulated fructose or an artificial sweetener (e.g., Sweet-n-Low®,NutraSweet®, or Equal®) and B. coagulans in spore form, freeze-driedvegetative cell form, or a combination thereof. Other artificialsweeteners include acesulfame K, aspartame, sucralose, d-tagatose, andcombinations thereof.

The probiotic organisms described herein, e.g., Bacillus coagulansstrain GBI-30 or BC³⁰, ATCC Designation Number PTA-6086, uniquelysurvive the harsh manufacturing and cooking processes of theconfection-based compositions. The confection-based compositions areprocessed for packaging by separating the confection-based compositionsfrom starch (e.g., corn starch). The confection-based compositions areheated to about 100° C. to make them “sticky”. Subsequently, theconfection-based compositions are placed in a drum tumbler, wherein theprobiotic/sugar coating is applied. Bacillus coagulans is blended withsugar prior to application to the surface of the confection-basedcomposition. The dry mix for confection-based compositions comprisessugar and an isolated Bacillus coagulans bacterium. The dry mix isbetween 1% and 50% Bacillus coagulans bacterium, e.g., about 5%, about10%, about 15%, about 20%, about 25%, about 35%, about 45%, or about 50%Bacillus coagulans bacterium. Preferably, the dry mix is about 15%Bacillus coagulans bacterium and 85% sugar. For example, about 100pounds of dry mix contains about 15 pounds of Bacillus coagulansbacterium and about 85 pounds of sugar.

The dry mix is between about 1% and about 50% by weight of theconfection-based composition, e.g., about 1% to about 20%, about 5% toabout 15%; about 6%, about 7%, about 8%, about 9%, or about 10% byweight of the confection-based composition. For example, a 3 gramconfection-based composition contains about 7% dry mix by weight of theconfection-based composition. A 3.8 to 4 gram confection-basedcomposition contains about 8-9% dry mix by weight of theconfection-based composition.

Alternatively, the isolated Bacillus coagulans bacterium is addeddirectly to the confection ingredients prior to heating, molding, andsubsequent cooling of the confection. In this manner, the probiotic isintroduced into the confection itself, instead of on the surface of theconfection-based composition.

As the recommended dietary allowances (RDA or recommended daily intake;RDI) is about 1×10⁹ bacterium (according to EU guidelines), preferably,the confection-based composition comprises at least about 1×10⁹ viablebacteria. In another aspect, the confection-based composition comprisesat least about 1×10⁶ to 1×10⁷; at least about 1×10⁷ to 1×10⁸; or atleast about 1×10⁸ to 1×10⁹ viable bacteria.

Probiotic Seafood Oil-Based Compositions

The invention is also directed to the surprising discovery that lacticacid-producing bacteria, particularly Bacillus species, remain viableand retain their beneficial probiotic properties in seafood/fishoil-based compositions. By “seafood” is meant any fish or shellfish fromthe sea used for food. Specifically, the probiotic organisms describedherein, e.g., Bacillus coagulans strain GBI-30 or BC³⁰, ATCC DesignationNumber PTA-6086, survive in the fish oil-based compositions describedbelow. The seafood/fish oil-based compositions are packaged insoft-shelled capsules or soft gelatin capsules or in the form ofsugar/gelatin (gummi) confections, e.g., both the Bacillus coagulansspores or bacterium and the fish oil are encapsulated together. Forexample, the fish oil and bacterial spores are incorporated intoconfection-based compositions, such as the gummi confections describedherein. Preferably, the bacterium is present as at least 90% spores,e.g., 95%, 98%, or 99% spores. The fish oil-based compositions aresuitable for human or animal consumption. In one aspect, the fishoil-based compositions are administered to children under 18 years ofage, e.g., under 15 years of age, under 10 years of age, or under 5years of age. Alternatively, the fish oil-based compositions areadministered to children and adults of all ages.

Fish oil contains two omega-3 fatty acids: eicosapentaenoic acid (EPA;all-cis-5,8,11,14,17-eicosapentaenoic acid) and docosahexaenoic acid(DHA; all-cis-4,7,10,13,16,19-docosahexaenoic acid). Omega-3 fatty acids(n-3 fatty acids or ω-3 fatty acids) are a family of unsaturated fattyacids that have a carbon-carbon double bond at the n-3 position, e.g.,at the third carbon bond from the terminal methyl end (n) of the fattyacid. Although fish are a dietary source of omega-3 fatty acids, fish donot produce the fatty acids themselves. Instead, omega-3 fatty acids,such as EPA and DHA are synthesized by microalgae and plankton that livein seawater. Fish accumulate omega-3 fatty acids by either consuming themicroalgae that produce the fatty acids, or by eating smaller prey fishthat have consumed the omega-3 fatty acids found in microalgae. Thus,fatty predatory fish like mackerel, lake trout, flounder, albacore tunaand salmon possess high levels of omega-3 fatty acids. Krill oil is alsoa source of omega-3 fatty acids.

The process by which oil is extracted from fish begins with cooking thefish product through a process of steam heating, wherein the fish willreach a top temperature of almost 100° C. This important step not onlysterilizes the fish, but also causes the proteins to coagulate and thealteration of cell membranes to aid in the extraction of the oil fromthe dry material. In some cases, the raw fish is hashed (cut intopieces) prior to steam cooking. After cooking, the mass of fish ispressed or centrifuged to separate the fat-free dry solids (mass offish) from the liquid (oil & water). This process also creates a fishpresscake, which is used by many facilities for the production of fishmeal commonly used in animal feed. The liquid collected from mass offish contains not only water, but also fish oil, salts, proteins, andeven undesired waste particles and toxins. The liquid (oil & water) isfurther filtered to separate the oil and water. At this point, theunrefined fish oil (also referred to as crude fish oil) has notundergone any portion of the refining process.

When fish oil is extracted from fish, so too are the free fatty acidsand toxins that are present in the fish. In some cases, fish oils arerefined and processed to remove impurities from the fish oil and toenhance the fatty acid potency. Ultra-refined fish oil has been throughsophisticated and intensive filtering and refining processes (e.g.,winterization) to produce pure and concentrated oil that is as far aspossible, free from contaminants. During winterization, the oil ischilled to allow filtration of the saturated fats and particles thatform at colder temperatures. Mercury and other metals are subsequentlyremoved before the oil is converted to ethyl esters, subjected totrans-esterification, and molecular or vacuum distillation to removeother fats and undesirable elements and to concentrate the oil.Optionally, fish oil is combined with preservatives and otheringredients suitable for mammalian consumption. For example, in somecases, acid clay is added to remove the pungent smell from the fish oil.

The omega-3 fatty acids derived from the tissues of oily fish, e.g.,salmon, herring, anchovies, sardines, tuna, pollock, cod, catfish,flounder, grouper, halibut, mahi mahi, orange roughy, red snapper,shark, swordfish, tilefish, and king mackerel have many health benefits.For example, the omega-3 fatty acids found in fish oil reduceinflammation, slow the spread of cancerous tissue, regulate cholesterollevels, improve cardiovascular health, boost the immune system, andprotect the brain from a variety of disorders, such as clinicaldepression, anxiety, Alzheimer's disease, and Parkinson's disease.

The fish oil-based and confection-based compositions are formulated inmany configurations, because the bacterium is present as a vegetativecell or as a spore, or both, depending on the species and form of theprobiotic organism. The cells/spores are formulated in a variety ofcompositions suited for use in a fish oil-based or confection-basedcomposition. In one aspect, the bacterium is present as a mixture ofspores and vegetative cells. In another aspect, the bacterium is presentas at least 90% spores, e.g., 95%, 98%, or 99% spores. Optionally, priorto addition to the fish oil-based or confection-based compositions ofthe invention, the Bacillus coagulans cells are cultured in liquid inthe absence of or with limited quantities of a food source to inducesporulation. In another aspect, heat gun spray drying kills about 50%,about 75%, about 90%, about 95%, or about 99% of vegetative cells priorto addition to the fish oil-based or confection-based compositions ofthe invention.

In one aspect, Bacillus coagulans bacteria in the form of a spray-driedpowder is included in or on the surface of the confection-basedcomposition described herein. Preferably, the isolated Bacilluscoagulans is in the form of a spore. The isolated Bacillus coagulans areat least 85%, at least 90%, at least 95%, or at least 99% pure spores.Alternatively, the isolated Bacillus coagulans is in the form of avegetative cell. In one aspect, the isolated Bacillus coagulans are atleast 85%, at least 90%, or at least 95% pure vegetative cells. Inanother aspect, the isolated Bacillus coagulans is in the form of amixture of vegetative cells and spores. The Bacillus coagulans mixtureis 90% spores, 10% vegetative cells; 75% spores, 25% vegetative cells;60% spores, 40% vegetative cells; 50% spores, 50% vegetative cells; 60%vegetative cells, 40% spores; 75% vegetative cells; 25% spores; or 90%vegetative cells, 10% spores.

The Bacillus and/or Bacillus coagulans is applied using any of a varietyof known methods including, for example, applying a powder, spray-dryingthe probiotic onto the confection-based composition, or soaking thecomposition in a solution containing the probiotic. Alternatively, theBacillus bacterium is mixed with the confection ingredients (e.g., gummiingredients) prior to boiling in water.

Any of a variety of methods for placing the bacterial composition into afish oil-based or confection-based composition can be used. In oneaspect, a “spray-dry” method is used, in which the compositions areexposed in a low humidity chamber to an atomized mix containing a liquidcomposition, where the chamber is subsequently exposed to approximately80-110° F. to dry the liquid, thereby impregnating the material of fishoil-based or confection-based composition with the components.

A typical concentration is from approximately 1×10⁷ to 1×10¹² CFU; 1×10⁸to 1×10¹¹ CFU; or 1×10⁹ to 1×10¹⁰ CFU of viable bacterium or spores/g offish oil or confection matrix or sanding sugar. Sanding sugar comprisescane sugar, beet sugar, date sugar, sucanat, granulated fructose or anartificial sweetener (e.g., Sweet-n-Low®, NutraSweet®, or Equal®) and B.coagulans in spore form, freeze-dried vegetative cell form, or acombination thereof. Following drying, the fish oil-based composition orconfection is ready for immediate use or for storage in a sterilepackage, e.g., a 3-ounce package (e.g., a bag or a bottle), a 6-ouncepackage, a 9-ounce package, a 12-ounce package, a 15-ounce package, an18-ounce package, or a 24-ounce package.

The active ingredients (i.e., live bacteria or extracellularcomponents), comprise between about 0.01% to about 10%; 0.01% to about1%; or about 0.05% to about 0.1% by weight of the probiotic fishoil-based or confection-based composition. Optionally, the isolatedBacillus coagulans comprise about 1 mg to about 10 g; about 10 mg toabout 1 g; or about 25 mg to about 75 mg by weight of the probioticcomposition. Most preferably, the amount of Bacillus coagulans bacteriais about 5×10⁷ colony forming units (CFU) of bacteria per gram of foodmatrix.

In one aspect, the amount of bacteria is about 10⁴ to 10¹⁴ colonyforming units (CFU) of bacteria per gram of probiotic composition (i.e.,vegetative cells and/or bacterial spores), preferably 10⁵ to 10¹³ CFU/gof fish oil or confection matrix. Alternatively, the concentrations are10⁸ to 10¹³ CFU/g; 10⁹ to 10¹² CFU/g; or 10¹⁰ to 10¹¹ CFU/g of fish oilor confection matrix. In one aspect, the amount of bacteria is about1×10⁶ CFU per gram of fish oil or confection matrix. The actual amountin a fish oil-based or confection-based composition will vary dependingupon the amounts of composition to be dispersed into the fish oil orconfection composition and upon routes of dispersal.

In one aspect, the invention provides for storing the fish oil-based orconfection-based composition in a sterile package at room temperatureprior to consumption. Alternatively, the composition is consumedimmediately.

By way of example, and not of limitation, Bacillus coagulans spores areincorporated into any type of dry or lyophilized product which isdissolved or mixed with hot water, so long as the temperature of theBacillus coagulans spore-containing mixture is raised to the requiredheat-shock temperature (i.e., 80° C. for 5 minutes) necessary forgermination of the spores. The Bacillus coagulans spores may either beincorporated into the dry or lyophilized product by the manufacturer ofthe product or by the consumer during preparation. The fish oil-based orconfection-based composition is subsequently boiled in hot water, e.g.,stovetop boiling, addition of boiling water to a container, ormicrowaving the fish oil-based or confection-based composition alongwith water.

The Bacillus coagulans spores survive storage (shelf-life), i.e., retainviability or the ability to germinate at physiological conditions (e.g.,ingestion), from about 12 days to about 2 years; from about 1 month toabout 18 months; from about 3 months to about 1 year; or from about 6months to about 9 months.

Example 1 Preparation of Bacillus Coagulans Cultures

Bacillus coagulans Hammer bacteria (ATCC Accession No. 31284) wasinoculated and grown to a cell density of about 10⁸ to 10⁹ cells/ml innutrient broth containing 5 g Peptone, 3 g Meat extract, 10-30 mg MnSO₄,and 1,000 ml distilled water, adjusted to pH 7.0, using a standardairlift fermentation vessel at 30° C. The range of MnSO₄ acceptable forsporulation is 1 mg/l to 1 g/l. The vegetative cells can activelyreproduce up to 45° C., and the spores are stable up to 90° C. Afterfermentation, the B. coagulans bacterial cells or spores are collectedusing standard methods (e.g., filtration, centrifugation) and thecollected cells and spores can be lyophilized, spray-dried, air-dried orfrozen. The supernatant from the cell culture is collected and used asan extracellular agent secreted by B. coagulans.

A typical yield from the above culture is in the range of about 10⁹ to10¹⁰ viable spores and more typically about 100 to 150 billioncells/spores per gram before drying. Spores maintain at least 90%viability after drying when stored at room temperature for up to tenyears, and thus the effective shelf life of a composition containing B.coagulans Hammer spores at room temperature is about 10 years.

Example 2 Preparation of Bacillus Coagulans Spores

A culture of dried B. coagulans spores was prepared as follows. Tenmillion spores were inoculated into a one liter culture containing 24 gpotato dextrose broth, 10 g of enzymic-digest of poultry and fishtissue, 5 g of FOS and 10 g MnSO₄. The culture was maintained for 72hours under a high oxygen environment at 37° C. to produce culturehaving about 150 billion cells per gram of culture. Thereafter, theculture was filtered to remove culture medium liquid, and the bacterialpellet was resuspended in water and freeze-dried. The freeze-driedpowder is then ground to a fine powder using standard good manufacturingpractice (GMP).

Example 3 Bacillus Coagulans Spores Survive in the Gastric Environment

This study was performed in order to determine the survivability rate ofBacillus coagulans spores as they pass through the stomach. Samples ofBacillus coagulans spores were subjected to a simulated gastricenvironment for varying lengths of time in order to attain theirsurvivability rate. First, a homogeneous sample of raw material Bacilluscoagulans of at least 12 grams was prepared. Saline solution at pH 1 wasprepared using 3N HCl (150 mls each into six 250 ml media bottles) andsterilized. Additional saline solutions with pH 2 and 3 were preparedsimilarly, resulting in 6 sterile 250 ml bottles, each containing 150 mlpH adjusted saline. Six sterile 250 ml media bottles each containing 150ml normal saline solution were prepared and sterilized. Phosphate buffer(˜400 ml) was prepared at pH 7.2. Test tubes (24) were prepared andsterilized, each containing 9 ml of phosphate buffer pH 7.2. Test tubes(120) were prepared, each containing 9 ml of normal saline. GYE(glucose-yeast extract) agar medium was prepared and sterilized andcooled to 45° C. in a water bath. Samples (24) of raw material wereweighed, each ˜500 milligrams (theoretically equivalent to 10 billionspores). The samples were added to media bottles at 37° C. and incubatedhalf for 20 minutes the other half for 120 minutes. After 20 and 120minutes incubation, respectively, the samples were mixed to uniformityand pipet 1 ml into 9 ml of sterile phosphate buffer pH 7.2. After all12 samples from each time point were placed into test tubes containingsterile phosphate buffer, serial dilutions were made until 6 tubes hadbeen used for each sample. The final dilution for the final two testtubes were 3×10⁷ and 3×10⁸, which gave a count of roughly 300 and 30CFU, respectively. The final 2 test tubes from each sample were placedinto 70° C. water bath for 30 minutes. After 30 minutes, they werecooled immediately to 45° C. Three sterile petri plates per tube wereset out. 1.0 ml from the heat-treated tube was added into each petriplate, then 15 ml of sterile molten GYE Agar medium (at 45° C.) waspoured into each of the petri plates and mixed thoroughly. Whensolidified, the plates were incubated in an inverted position for 48hours at 40° C. The individual colonies were counted. Results wereexpressed as CFU per gram as shown in Table 1 below. 1.0E+10=1×10¹⁰.

TABLE 1 20 Minutes 120 Minutes Incubation Incubation Spore Count, SporeCount, Sample CFU/gram CFU/gram Normal Saline - A 1.90E+10 1.88E+10Normal Saline - B 2.12E+10 2.00E+10 Normal Saline - C 1.64E+10 2.06E+10Average 1.89E+10 1.98E+10 Saline pH 1.0 - D 2.08E+09 5.98E+07 Saline pH1.0 - E 1.47E+09 0.00E+00 Saline pH 1.0 - F 3.59E+09 0.00E+00 Average2.38E+09 1.99E+07 Saline pH 2.0 - G 3.63E+09 3.46E+09 Saline pH 2.0 - H4.47E+09 2.48E+09 Saline pH 2.0 - I 3.58E+09 2.82E+09 Average 3.89E+092.92E+09 Saline pH 3.0 - J 1.65E+10 1.13E+10 Saline pH 3.0 - K 1.35E+101.11E+10 Saline pH 3.0 - L 1.80E+10 1.39E+10 Average 1.60E+10 1.21E+10

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. Genbank and NCBI submissions indicated byaccession number cited herein are hereby incorporated by reference. Allother published references, documents, manuscripts and scientificliterature cited herein are hereby incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1-25. (canceled)
 26. A composition comprising an omega-3 fatty acid andisolated Bacillus coagulans spores.
 27. The composition of claim 26,wherein the omega-3 fatty acid comprises eicosapentaenoic acid ordocosahexaenoic acid.
 28. The composition of claim 26, wherein saidomega-3 fatty acid has been produced by microalgae.
 29. The compositionof claim 26, wherein the omega-3 fatty acid is in oil.
 30. Thecomposition of claim 29, wherein the oil comprises seafood oil.
 31. Thecomposition of claim 29, wherein the oil comprises shellfish oil or fishoil.
 32. The composition of claim 29, wherein the oil comprises hilloil.
 33. The composition of claim 29, wherein the oil comprises salmonoil, cod oil, herring oil, anchovy oil, sardine oil, or pollock oil,tuna oil, catfish oil, flounder oil, lake trout oil, grouper oil,halibut oil, mahi mahi oil, orange roughy oil, red snapper oil, sharkoil, swordfish oil, tilefish oil, or mackerel oil.
 34. The compositionof claim 29, wherein the oil comprises cod oil, wherein the cod oil iscod liver oil.
 35. The composition of claim 26, wherein said isolatedBacillus coagulans spores are coated on the exterior surface of saidcomposition.
 36. The composition of claim 26, comprising a confection.37. The composition of claim 36, wherein said composition comprises agranulated or powder sugar coating on the exterior thereof, wherein saidcoating comprises said isolated Bacillus coagulans spores.
 38. Thecomposition of claim 36, wherein the confection comprises a gummiconfection.
 39. The composition of claim 36, wherein the confectioncomprises a hard sweet, fudge, toffee, liquorice, chocolate, jellycandy, a marshmallow, or marzipan.
 40. The composition of claim 26,wherein said composition is encapsulated in a soft-shelled capsule or asoft gelatin capsule.
 41. The composition of claim 31, comprising fishoil, wherein the fish oil has been processed to remove an impurity. 42.The composition of claim 41, wherein the impurity is a toxin.
 43. Thecomposition of claim 42, wherein the toxin is mercury.
 44. Thecomposition of claim 26, further comprising a preservative.
 45. Thecomposition of claim 26, wherein said isolated Bacillus coagulans sporescomprise between 0.01% to about 1% by weight of said composition. 46.The composition of claim 26, wherein said isolated Bacillus coagulansspores are GBI-30 strain (ATCC Designation Number PTA-6086) spores,GBI-20 strain (ATCC Designation Number PTA-6085) spores, or GBI-40strain (ATCC Designation Number PTA-6087) spores.
 47. The composition ofclaim 31, comprising fish oil and 1×10⁷ to 1×10¹² CFU; 1×10⁸ to1×10¹¹CFU; or 1×10⁹ to 1×10¹⁰ CFU of viable isolated Bacillus coagulansspores per gram of fish oil.
 48. The composition of claim 26, which isstored at room temperature.
 49. The composition of claim 48, whereinsaid isolated Bacillus coagulans spores retain viability or the abilityto germinate at physiological conditions, from about 12 days to about 2years; from about 1 month to about 18 months; from about 3 months toabout 1 year; or from about 6 months to about 9 months, when stored atroom temperature.
 50. A method of boosting the immune system, comprisingadministering to a subject the composition of claim
 26. 51. A method ofimproving cardiovascular health, comprising administering to a subjectthe composition of claim
 26. 52. A method of reducing inflammation,comprising administering to a subject the composition of claim
 26. 53. Amethod of protecting the brain from a disorder, comprising administeringto a subject the composition of claim 26.